1. Field of the Invention
The present invention relates to an enzyme for cleaving within the wall anchor region of a surface protein of a gram-positive bacterium, methods for detecting that enzyme in vivo and in vitro, and methods for purifying that enzyme. In particular, the enzyme is isolated from a group A Streptococcus, and cleaves the anchor region of streptococcal M protein.
2. Description of the Related Art
Bacteria may be designated as gram-positive and gram-negative, based on the staining of their cell walls with Gram's stain. Within the broad division of gram-positive bacteria are the gram-positive cocci, which include the genera Aerococcus, Corprococcus, Deinobacter, Deinococcus, Enterococcus, Gemella, Lactococcus, Leuconostoc, Marinococcus, Melissococcus, Micrococcus, Pediococcus, Peptococcus, Peptostreptococcus, Planococcus, Ruminococcus, Saccharococcus, Salinicoccus, Carcina, Staphylococcus, Stomatococcus, Streptococcus, Trichococcus, Vagococcus, Listeria and Actinomyces. In gram-positive bacteria, proteins are secreted into the surrounding medium, whereas in gram-negative bacteria, secretion occurs into the periplasmic space between the cytoplasmic and outer membranes (Model and Russel (1990) Cell 61:739-741; Schatz and Beckwith (1990) Ann. Rev. Genet. 24:215-248). Prokaryotic sorting signals are conceivable for the local assembly of supramolecular structures like pili (Strom and Lory, (1987) J. Bacteriol. 169:3181-3188), flagella (Loewy et al (1987) Genes Dev. 1:626-635), and bacteriophages (Brissette and Russel (1990) J. Mol. Biol. 211:565-580), or for localization of proteins in defined bacterial compartments. Such compartments include the outer membrane of gram-negative bacteria (Model and Russel (1990) Cell 61:739-741), the cell wall (Braun et al (1970) Eur. J. Biochem. 13:336-346; Eur. J. Biochem. 14:387-391; Biochemistry 9:5041-5049; Shockman and Barrett (1983) Annu. Rev. Microbiol. 37:501-527), and the periseptal annulus (MacAlister et al (1983) Proc. Natl. Acad. Sci. 80:1372-1376).
The cell wall of gram-positive bacteria can be thought of as representing a unique cell compartment, which contains anchored surface proteins that require specific sorting signals. Some biologically important products are anchored in this way, including protein A and fibronectin binding proteins of Staphylococcus aureus and M protein from Streptococcus pyogenes. Studies of staphylococcal Protein A and E. coli alkaline phosphatase show that the signal both necessary and sufficient for cell wall anchoring consists of an LPXTGX motif (SEQ ID NO:1), a C-terminal hydrophobic domain, and a charged tail. These sequence elements are conserved in many surface proteins from different gram-positive bacteria.
M protein of group A streptococci, an .alpha.-helical coiled-coil fibrillar molecule found on the surface of the organism (Fischetti et al (1988) Proteins Struct. Func. Genet. 3:60), is responsible for the antiphagocytic property of these bacteria (Lancefield et al (1962) J. Immunol. 89:307). Antigenic variation (Jones et al (1988), Proc. Natl. Acad. Sci. USA 85:8271) and type-specific immunity are contingent upon epitopes located within the NH.sub.2 -terminal half of the M molecule (distal to the cell wall) (Jones et al (1988) J. Exp. Med. 167:1114). Amino acid sequences that are conserved among different M proteins are located in the COOH-terminal half (Jones et al (1985) J. Exp. Med. 161:623; Hollingshead et al (1987) Infect. Immun. 55:3237) and contain epitopes recently shown to be responsible for non-type-specific immunity against streptococcal colonization (Bessen et al (1988) Infect. Immun. 56:2666; Fischetti et al (1989) Science 244:1487).
The attachment region of the molecule, predicted from DNA sequence, is located at the COOH terminal end, composed of charged amino acids at the COOH terminus, followed by 19 hydrophobic amino acids suspected to be a membrane anchor followed by a hexapeptide motif, LPXTGX. This region is adjacent to a proline and glycine-rich region situated within the peptidoglycan layer of the cell wall (Fischetti (1988) Proteins Struct. Func. Genet. 3:60; Pancholi et al (1988) J. Bacteriol. 170:2618; Hollingshead et al (1986), J. Biol. Chem. 261:1677.)
The association of the M protein to the cytoplasmic membrane of gram-positive bacteria can be examined after removing the cell wall with the muralytic enzyme lysin (Fischetti et al. (1974) Streptococcal Disease and the Community, M. J. Haverkorn, editor. Excerpta Medica, Amsterdam. 26.), which is active against group A streptococcal cell walls over a broad pH range. M protein is released during the removal of the cell wall indicating that an endogenous factor mediates this release (Pancholi et al (1989) J. Exp. Med. 170:2119-2133).
Analysis of the released form of the M protein demonstrates that the COOH-terminal 19 hydrophobic amino acids and charged tail of the M molecule are not present (Pancholi et al (1989) J. Exp. Med. 170:2119-2133). This suggests that the release of M proteins from the membrane and its attachment to the cell wall is in some way associated with the cleavage of the COOH-terminal hydrophobic region.
Cleavage of the surface proteins of gram-positive bacteria in the LPXTGX region adjacent to the hydrophobic domain has been shown to occur during the anchoring process of these proteins (Schneewind et al (1992) Cell 70:267-281). Because interference with this cleavage prevents the proper placement of surface proteins on the bacterial cell, characterization of the enzyme responsible for this cleavage would be a critical step in antibiotic development.
Therefore, it is apparent that what is needed in this art is a method of detecting and isolating this gram-positive bacterial surface protein cleavage enzyme.